Genomic Autopsy of Sudden Deaths in Young Individuals.

Importance: Postmortem genetic testing of young individuals with sudden death has previously identified pathogenic gene variants. However, prior studies primarily considered highly penetrant monogenic variants, often without detailed decedent and family clinical information. Objective: To assess genotype and phenotype risk in a diverse cohort of young decedents with sudden death and their families. Design, Setting, and Participants: Pathological and whole-genome sequence analysis was conducted in a cohort referred from a national network of medical examiners. Cases were accrued prospectively from May 2015 to March 2019 across 24 US states. Analysis began September 2016 and ended November 2020. Exposures: Evaluation of autopsy and clinical data integrated with whole-genome sequence data and family member evaluation. Results: A total of 103 decedents (mean [SD] age at death, 23.7 [11.9] years; age range, 1-44 years), their surviving family members, and 140 sex- and genetic ancestry-matched controls were analyzed. Among 103 decedents, autopsy and clinical data review categorized 36 decedents with postmortem diagnoses, 23 decedents with findings of uncertain significance, and 44 with sudden unexplained death. Pathogenic/likely pathogenic (P/LP) genetic variants in arrhythmia or cardiomyopathy genes were identified in 13 decedents (12.6%). A multivariable analysis including decedent phenotype, ancestry, and sex demonstrated that younger decedents had a higher burden of P/LP variants and select variants of uncertain significance (effect size, -1.64; P = .001). These select, curated variants of uncertain significance in cardiac genes were more common in decedents than controls (83 of 103 decedents [86%] vs 100 of 140 controls [71%]; P = .005), and decedents harbored more rare cardiac variants than controls (2.3 variants per individual vs 1.8 in controls; P = .006). Genetic testing of 31 parent-decedent trios and 14 parent-decedent dyads revealed 8 transmitted P/LP variants and 1 de novo P/LP variant. Incomplete penetrance was present in 6 of 8 parents who transmitted a P/LP variant. Conclusions and Relevance: Whole-genome sequencing effectively identified P/LP variants in cases of sudden death in young individuals, implicating both arrhythmia and cardiomyopathy genes. Genomic analyses and familial phenotype association suggest potentially additive, oligogenic risk mechanisms for sudden death in this cohort.

Mitochondrial cardiomyopathy and ventricular arrhythmias associated with biallelic variants in C1QBP.

Patients with biallelic mutations in the nuclear-encoded mitochondrial gene C1QBP/p32 have been described with syndromic features and autosomal recessive cardiomyopathy. We describe the clinical course in two siblings who developed cardiomyopathy and ventricular fibrillation in infancy. We provide genomic analysis and clinical-pathologic correlation. Both siblings had profound cardiac failure with ventricular arrhythmia. One child died suddenly. The second sibling survived resuscitation but required extracorporeal cardiopulmonary support and died shortly afterward. On cardiac autopsy, the left ventricle was hypertrophied in both children. Histological examination revealed prominent cardiomyocyte cytoplasmic clearing, and electron microscopy confirmed abnormal mitochondrial structure within cardiomyocytes. DNA sequencing revealed compound heterozygous variants in C1QBP (p.Thr40Asnfs*45 and p.Phe204Leu) in both children. Family segregation analysis demonstrated each variant was inherited from an unaffected, heterozygous parent. Inherited loss of C1QBP/p32 is associated with recessive cardiomyopathy, ventricular fibrillation, and sudden death in early life. Ultrastructural mitochondrial evaluation in the second child was similar to findings in engineered C1qbp-deficient mice. Rapid trio analysis can define rare biallelic variants in genes that may be implicated in sudden death and facilitate medical management and family planning. (184/200).

Transcriptional analysis of histone deacetylase family members reveal similarities between differentiating and aging spermatogonial stem cells.

The differentiation of adult stem cells involves extensive chromatin remodeling, mediated in part by the gene products of histone deacetylase (HDAC) family members. While the transcriptional downregulation of HDACs can impede stem cell self-renewal in certain contexts, it may also promote stem cell maintenance under other circumstances. In self-renewing, differentiating, and aging spermatogonial stem cells (SSCs), the gene expression dynamics of HDACs have not yet been characterized. To gain further insight with these studies, we analyzed the transcriptional profiles of six HDAC family members, previously identified to be the most highly expressed in self-renewing SSCs, during stem cell differentiation and aging. Here we discovered that in both differentiating and aging SSCs the expression of Sirt4 increases, while the expression of Hdac2, Hdac6, and Sirt1 decreases. When SSCs are exposed to the lifespan-enhancing drug rapamycin in vivo, the resultant HDAC gene expression patterns are opposite of those seen in the differentiating and aging SSCs, with increased Hdac2, Hdac6, and Sirt1 and decreased Hdac8, Hdac9, and Sirt4. Our findings suggest that HDACs important for stem cell maintenance and oxidative capacity are downregulated as adult stem cells differentiate or age. These results provide important insights into the epigenetic regulation of stem cell differentiation and aging in mammals.

Distinct requirements for Sin3a in perinatal male gonocytes and differentiating spermatogonia.

Chromatin modifier Swi-independent 3a (SIN3A), together with associated histone deacetylases, influences gene expression during development and differentiation through a variety of transcription factors in a cell-specific manner. Sin3a is essential for the maintenance of inner cell mass cells of mouse blastocysts, embryonic fibroblasts, and myoblasts, but is not required for the survival of trophectoderm or Sertoli cells. To better understand how this transcriptional regulator modulates cells at different developmental stages within a single lineage, we used conditional gene targeting in mice to ablate Sin3a from perinatal quiescent male gonocytes and from postnatal differentiating spermatogonia. Mitotic germ cells expressing stimulated by retinoic acid gene 8 (Stra8) that lacked Sin3a exhibited increased DNA damage and apoptosis, yet collectively progressed through meiosis and spermiogenesis and generated epididymal sperm at approximately 50% of control levels, sufficient for normal fertility. In contrast, perinatal gonocytes lacking Sin3a underwent rapid depletion that coincided with cell cycle reentry, exhibiting 2.5-fold increased histone H3 phosphorylation upon cycling that suggested a prophase/metaphase block; germ cells were almost entirely absent two weeks after birth, resulting in sterility. Gene expression profiling of neonatal testes containing Sin3a-deleted gonocytes identified upregulated transcripts highly associated with developmental processes and pattern formation, and downregulated transcripts involved in nuclear receptor activity, including Nr4a1 (Nur77). Interestingly, Nr4a1 levels were elevated in testes containing Stra8-expressing, Sin3a-deleted spermatogonia. SIN3A directly binds to the Nr4a1 promoter, and Nr4a1 expression is diminished upon spermatogonial differentiation in vitro. We conclude that within the male germline, Sin3a is required for the mitotic reentry of gonocytes, but is dispensable for the maintenance of differentiating spermatogonia and subsequent spermatogenic processes.

Rapamycin increases oxidative stress response gene expression in adult stem cells.

Balancing quiescence with proliferation is of paramount importance for adult stem cells in order to avoid hyperproliferation and cell depletion. In some models, stem cell exhaustion may be reversed with the drug rapamycin, which was shown can suppress cellular senescencein vitro and extend lifespan in animals. We hypothesized that rapamycin increases the expression of oxidative stress response genes in adult stem cells, and that these gene activities diminish with age. To test our hypothesis, we exposed mice to rapamycin and then examined the transcriptome of their spermatogonial stem cells (SSCs). Gene expression microarray analysis revealed that numerous oxidative stress response genes were upregulated upon rapamycin treatment, including superoxide dismutase 1, glutathione reductase, and delta-aminolevulinate dehydratase. When we examined the expression of these genes in 55-week-old wild type SSCs, their levels were significantly reduced compared to 3-week-old SSCs, suggesting that their downregulation is coincident with the aging process in adult stem cells. We conclude that rapamycin-induced stimulation of oxidative stress response genes may promote cellular longevity in SSCs, while a decline in gene expression in aged stem cells could reflect the SSCs' diminished potential to alleviate oxidative stress, a hallmark of aging.